Note: Descriptions are shown in the official language in which they were submitted.
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ANTHELMINTIC IMIDAZOL-THIAZOLE DERIVATES
The present invention is concerned with novel anthelmintic tetramisole
derivatives and
the pharmaceutically acceptable acid addition salts thereof, compositions
comprising
said novel compounds, processes for preparing said compounds and compositions,
and
the use thereof as a medicine, in particular in treatment, control and
prevention of endo-
and ectoparasite infections in warm-blooded animals.
Tetramisole and levamisole are very well known anthelmintics having the
following
structure :
N
(tetramisole is the racemic dl-form, levamisole is the enantiomeric pure 1-
form).
One of the most popular anthelmintics is levamisole which has been widely used
to
control nematode parasites in farm animals, in particular in sheep and cattle.
However
the development of nematodes resistant to anthelmintics has become a major
problem
in farm animals, particularly with the nematode Haemonchus contortus. Moreover
multi-resistant strains have been found in field circumstances that have
developed
resistance against widely used anthelmintics such as levamisole, mebendazole
and
ivermectin. Hence there is a need to fmd new anthelmintic agents having
anthelmintic
activity against levamisole resistant and multi-resistant nematodes.
Other tetramisole anthelmintics have been disclosed in, e.g. US-4,014,892
which
exemplifies furan-2-carboxylic acid [3-(2,3,5,6-tetrahydro-imidazo[2,1-
b]thiazol-6-yl)-
phenyl]-amide as compound (163) and (tetrahydrofuran-2-ylmethyl)-[3-(2,3,5,6-
tetrahydro-imidazo[2,1-b]thiazol-6-yl)-phenyl]-amine as compound (110). The
latter
compound is also disclosed in GB-1,365,515 as compound (81).
The present invention relates to novel compounds of formula (I)
P0-C N
H
(1)
0
the pharmaceutically acceptable acid addition salts and the stereochemically
isomeric
forms thereof.
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As demonstrated in pharmacological example C.1 the compounds of the present
invention have an unexpected better anthelmintic activity against a multi-
resistant
strain of Haemonchus contortus (resistant against levamisole, mebendazole,
ivermectin
and closantel) than the art known compounds furan-2-carboxylic acid [3-
(2,3,5,6-
tetrahydro-imidazo[2,1-b]thiazol-6-yl)-phenyl]-amide (referenced to as
compound (A)
in the description) and (tetrahydrofuran-2-ylmethyl)-[3-(2,3,5,6-tetrahydro-
imidazo[2,1-b]thiazol-6-yl)-phenyl]-amine (referenced to as compound (B) in
the
description).
The pharmaceutically acceptable acid addition salts as mentioned hereinabove
are
meant to comprise the therapeutically active non-toxic acid addition salt
forms that the
compounds of formula (I) are able to form. These pharmaceutically acceptable
acid
addition salts can conveniently be obtained by treating the base form with
such
appropriate acid. Appropriate acids comprise, for example, inorganic acids
such as
hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric,
phosphoric and
the like acids; or organic acids such as, for example, acetic, propanoic,
hydroxyacetic,
lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e.
butanedioic acid),
maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic,
benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic,
pamoic and
the like acids.
Conversely said salt forms can be converted by treatment with an appropriate
base into
the free base form.
The term "stereochemically isomeric forms" as used hereinbefore defines all
the
possible isomeric forms which the compounds of formula (I) may possess. Unless
otherwise mentioned or indicated, the chemical designation of compounds of
formula
(I) denotes the mixture of all possible stereochemically isomeric forms, said
mixtures
containing all diastereomers and enantiomers of the basic molecular structure.
More in
particular, stereogenic centers may have the R- or S-configuration. The
compounds of
formula (I) have two chiral carbon atoms, as indicated by an asterisk in the
structure
below
P0_* NNI~
H(1)
O
)/-*a
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giving a total of 4 different stereoisomers. A114 individual stereochemically
isomeric
forms of the compounds of formula (I), and every possible mixture thereof, are
obviously intended to be embraced within the scope of this invention.
The absolute stereochemical configuration of the compounds of formula (I) and
of the
intermediates used in their preparation may easily be determined by those
skilled in the
art while using well-known methods such as, for example, X-ray diffraction.
Furthermore, some compounds of formula (I) and some of the intermediates used
in
their preparation may exhibit polymorphism. It is to be understood that the
present
invention encompasses any polymorphic forms possessing properties useful in
the
treatment of the conditions noted hereinabove.
A particular group of compounds are those compounds of formula (I-a) which are
defined as compounds of formula (I) having the (S)-configuration at the 6-
position of
the 2,3,5,6-tetrahydro-imidazo[2,1-b]thiazole moiety.
/ HN 0 0 (I-a)
A preferred compound is (2R)-tetrahydro-furan-2-carboxylic acid (6S)-[3-
(2,3,5,6-
tetrahydro-imidazo[2,1-b]thiazol-6-yl)-phenyl]-amide.
Compounds of formula (I) can in general be prepared by reacting an
intermediate of
formula (V) dissolved in a dioxane solution acidified with HC1 with an
intermediate of
formula (IV) in a suitable reaction-inert solvent such as acetonitrile.
C1 +
a-r N S
O H2N
intm. (V) intm. (IV)
Intermediates of formula (IV) can be prepared as outlined below. The
commercially
available starting compound 2-bromo-1-(3-nitrophenyl)-ethanone acid is reacted
with
4,5-dihydrothiazolamine and the obtained intermediate (I) is then reduced with
an
appropriate reducing agent such as sodium borohydride in a suitable solvent
such as
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ethanol yielding intermediate (II). Intermediate (II) is then treated with
thionyl chloride
in a reaction-inert solvent such as dichloroethane whereby the 2,3,5,6-
tetrahydro-
imidazo[2,1-b]thiazolyl ring of intermediate (III) is formed. Intermediate
(III) is first
converted into its HC1 addition salt before its nitro group is reduced to an
amino group
using art-known reduction agents such as iron powder and aqueous NH4C1 or
SnC12
thereby yielding intermediate (IV).
O 2 OHN ~S
02N Br N~ S 02N N~
reduction
intm. (I)
HOHN'~r S
02N NJ 1) SOC12 ~_c
S salt conversion
2) NaHC03/H20 Nv
intm. (II) 02N intm. (III)
S Fe / NH4C1
~J H20 / CH3OH
.HCI
02N H2N
intm. (III-HC1-salt) intm. (IV)
Intermediate (V) is prepared by converting tetrahydo-2-furancarboxylic acid
into its
acyl chloride analogue by reacting it with oxalylchloride. Intermediate (V)
can also be
prepared as its stereoisomeric pure (R)- or (S)-enantiomer by starting from
either (R)-
or (S)- tetrahydo-2-furancarboxylic acid respectively.
O O
OH Cl
a-Ir Cl~ Cl O
O
intm. (V)
The intermediate of formula (III) can be separated into its (+)- or (-)-
stereoisomers
using art-known separation techniques such as liquid chromatography using a
chiral
stationary phase.
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chiral N~ S separation (g) NZ_Z:: S
+ 02N intm. (III) 02N intm. (111-a) 02N intm. (III-b)
The compounds of formula (I) as prepared in the hereinabove described
processes may
be synthesized in the form of racemic mixtures of enantiomers which can be
separated
from one another following art-known resolution procedures. Those compounds of
formula (I) that are obtained in racemic form may be converted into the
corresponding
diastereomeric salt forms by reaction with a suitable chiral acid. Said
diastereomeric
salt forms are subsequently separated, for example, by selective or fractional
crystallization and the enantiomers are liberated therefrom by alkali. An
alternative
manner of separating the enantiomeric forms of the compounds of formula (I)
involves
liquid chromatography using a chiral stationary phase. Said pure
stereochemically
isomeric forms may also be derived from the corresponding pure
stereochemically
isomeric forms of the appropriate starting materials, provided that the
reaction occurs
stereospecifically. Preferably if a specific stereoisomer is desired, said
compound will
be synthesized by stereospecific methods of preparation. These methods will
advantageously employ enantiomerically pure starting materials.
The compounds of formula (I), the pharmaceutically acceptable salts and stereo-
isomeric forms thereof possess favorable anthelmintic activity. Therefore the
present
compounds of formula (I) are useful as a medicine in treatment, control and
prevention
of endo- and ectoparasite infections in warm-blooded animals.
Endo- and ectoparasites include Nemathelminthes such as Amidostomum,
Ancylostoma, Angiostrongylus, Anisakis, Ascaris, Brugia, Bunostomum,
Capillaria,
Chabertia, Cooperia, Cyathostomum, Cylicocyclus, Dictyocaulus (lungworm),
Dipetalonema, Dirofilaria (heartworm), Dracunculus, Elaeophora, Gaigeria,
Globocephalus urosubulatu, Haemonchus, Heterakis, Hyostrongylus,
Metastrongylus
(lungworm), Muellerius (lungworm), Necator americanus, Nematodirus,
Neoascaris,
Oesophagostomum, Onchocerca, Ostertagia, Oxyuris, Parascaris, Protostrongylus
(lungworm), Setaria, Stephanofilaria, Strongyloides, Strongylus, Syngamus,
Teladorsagia, Toxascaris, Toxocara, Trichinella, Trichostrongylus, Trichuris,
Uncinaria
stenocephala, and Wuchereria bancrofti.
Warm-blooded animals as used throughout this text include both human and non-
human animals such as farm animals (e.g. sheep, cattle, swine, goats or
horses),
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domestic animals (e.g. dogs, cats, or cavias) as well as wild animals held in
captivity
and birds (e.g. poultry).
In view of the utility of the compounds of formula (I), it follows that the
present
invention also provides a method of treating, controlling and preventing endo-
and
ecto-parasite infections in warm-blooded animals. This method comprises
administering to a warm-blooded animal in need thereof a therapeutically
effective
amount of a compound of formula (I).
The term "therapeutically effective amount of a compound of formula (I)" as
used
herein, means that amount of compound of formula (I) that elicits the
biological or
medicinal response in the warm-blooded animal that is being sought by the
physician or
veterinarian, which includes alleviation of the symptoms of the condition
being treated.
The therapeutically effective amount can be determined using routine
optimization
techniques and is dependent upon the particular condition to be treated, the
condition of
the warm-blooded animal, the route of administration, the formulation, and the
judgment of the practitioner and other factors evident to those skilled in the
art. A
therapeutically effective amount may be achieved by multiple dosing.
Additionally the present invention provides pharmaceutical compositions
comprising at
least one pharmaceutically acceptable carrier and a therapeutically effective
amount of
a compound of formula (I).
For use in warm-blooded animals, including humans, the compounds of formula
(I) can
be administered alone, but will generally be administered in admixture with a
pharmaceutically or veterinary acceptable diluent or carrier selected with
regard to the
intended route of administration and standard pharmaceutical practice. For
example,
they can be administered orally, including sublingually, in the form of
tablets
containing such excipients as starch or lactose, or in capsules or ovules
either alone or
in admixture with excipients, or in the form of elixirs, solutions or
suspensions
containing flavouring or colouring agents. The compounds of formula (I) could
be
incorporated into capsules, tablets or boluses for targeting the colon or
duodenum via
delayed dissolution of said capsules, tablets or boluses for a particular time
following
oral administration. The compounds of formula (I) can be injected
parenterally, for
example, intravenously, intramuscularly or subcutaneously. For parenteral
administration, they are best used in the form of a sterile aqueous solution
or
suspension that may contain other substances, for example, enough salt or
glucose to
make the solution isotonic with blood. The compounds of formula (I) can be
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administered topically, in the form of sterile creams, gels, pour-on or spot-
on
formulations, suspensions, lotions, ointments, dusting powders, sprays, drug-
incorporated dressings or via a skin patch. For example the compounds of
formula (I)
can be incorporated into a cream consisting of an aqueous or oily emulsion of
polyethylene glycols or liquid paraffin, or they can be incorporated into an
ointment
consisting of a white wax soft paraffin base, or as hydrogel with cellulose or
polyacrylate derivatives or other viscosity modifiers, or as a dry powder or
liquid spray
or aerosol with butane/propane, HFA or CFC propellants, or as a drug-
incorporated
dressing either as a tulle dressing, with white soft paraffm or polyethylene
glycols
impregnated gauze dressings or with hydrogel, hydrocolloid, alginate or film
dressings.
The compounds of formula (I) could also be administered intra-ocularly as an
eye drop
with appropriate buffers, viscosity modifiers (e.g. cellulose derivatives),
preservatives
(e.g. benzalkonium chloride (BZK)) and agents to adjust tenicity (e.g. sodium
chloride). Such formulation techniques are well-known in the art. All such
formulations may also contain appropriate stabilizers and preservatives.
For veterinary use, compounds can be administered as a suitably acceptable
formulation in accordance with normal veterinary practice and the veterinarian
will
determine the dosing regimen and route of administration which will be most
appropriate for a particular animal.
For topical application dip, spray, powder, dust, pour- on, spot-on,
emulsifiable
concentrate, jetting fluid, shampoos, collar, tag or harness may be used. Such
formulations are prepared in a conventional manner in accordance with standard
veterinary and pharmaceutical practice. Thus capsules, boluses or tablets may
be
prepared by mixing the active ingredient with a suitable finely divided
diluent or
carrier, additionally containing a disintegrating agent and/or binder such as
starch,
lactose, talc, or magnesium stearate. A drench formulation may be prepared by
dispersing the active ingredients in an aqueous solution together with
dispersing or
wetting agents and injectable formulations may be prepared in the form of a
sterile
solution or emulsion. Pour-on or spot-on formulations may be prepared by
dissolving
the active ingredients in an acceptable liquid carrier vehicle, such as butyl
digol, liquid
paraffin or non-volatile ester with or without addition of a volatile
component such as
isopropanol.
Alternatively, pour-on, spot-on or spray formulations can be prepared by
encapsulation
to leave a residue of active agent on the surface of the animal. These
formulations will
vary with regard to the weight of active compound depending on the species of
host
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animal to be treated, the severity and type of infection and type and body
weight of the
host. The formulations comprising a compound of formula (I) may be
administered
continuously, particularly for prophylaxis by known methods.
As an alternative the combinations may be administered with the animal
feedstuff and
for this purpose a concentrated feed additive or premix may be prepared for
mixing
with the normal animal feed.
For human use the compounds of formula (I) are administered as a
pharmaceutically
acceptable formulation in accordance with normal medical practice.
The compounds of formula (I) may be used in conjunction with other
anthelmintic or
antiparasitic agents so as to widen the spectrum of action or to prevent the
buildup of
resistance. Other anthelmintic agents are for example avermectines and
milbemycines
such as abamectin, cydectin, doramectin, eprinomectin, ivermectin, milbemycin,
milbemycin D, milbemycin oxime, moxidectin, selamectin, and the like;
benzimidazoles such as albendazole, cambendazole, fenbendazole, flubendazole,
mebendazole, oxfendazole, parbendazole, oxibendazole and cyclobendazole; pro-
benzimidazoles such as febantel, thiophanate and netobimin; salicylanilides
such as
closantel and niclosamide; imidazothiazoles such as butamisole and levamisole;
tetrahydropyrimidines such as morantel, pyrantel en pyrantel pamoate;
hexahydro-
pyrazinoisoquinolines such as praziquantel; and macrolides produced by
fermentation
of Saccharopolyspora spinosa such as spinosyn A, spinosyn D or spinosad.
Those skilled in the treatment of helminthiasis will easily determine the
therapeutically
effective amount of a compound of formula (I) from the test results presented
hereinafter. In general it is contemplated that a therapeutically effective
dose will be
from about 0.1 mg/kg to about 20 mg/kg of body weight, more preferably from
about
1 mg/kg to about 10 mg/kg of body weight of the warm-blooded animal to be
treated.
It may be appropriate to administer the therapeutically effective dose in the
form of two
or more sub-doses at appropriate intervals throughout the day.
The exact dosage and frequency of administration depends on the particular
compound
of formula (I) used, the particular condition being treated, the severity of
the condition
being treated, the age, weight and general physical condition of the
particular warm-
blooded animal as well as the other medication (including the above-mentioned
additional anthelmintic or antiparasitic agents), the warm-blooded may be
taking, as is
well known to those skilled in the art. Furthermore, said effective daily
amount may be
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lowered or increased depending on the response of the treated animal and/or
depending
on the evaluation of the physician or veterinarian prescribing the compounds
of the
instant invention. The effective daily amount ranges mentioned hereinabove are
therefore only guidelines.
Experimental part
A. Synthesis of the intermediates
Example A.1
C1
Preparation of 0~~~~~-= ~ intermediate (1)
(R)U
A solution of (+)-(R)-tetrahydo-2-furancarboxylic acid (6.65 ml) in
dichloromethane
(250 ml) was stirred under nitrogen at room temperature and then ethanedioyl
dichloride (12.1 ml) and anhydrous dimethylformamide (3 drops) were added. The
reaction mixture was stirred for 2.5 hours and then the solvent was evaporated
and co-
evaporated two times with toluene, yielding (2R)-tetrahydro-2-furancarbonyl
chloride
(intermediate 1).
In an analogous way racemic tetrahydro-2-furancarbonyl chloride was prepared
starting
from the commercially available tetrahydo-2-furancarboxylic acid and (2S)-
tetrahydro-
2-furancarbonyl chloride was prepared starting from commercially available (-)-
(S)-
tetrahydo-2-furancarboxylic acid.
Example A.2
HNS
a) Preparation of o2N intermediate (2)
A suspension of 4,5-dihydrothiazolamine (1.95 mol) in 2-propanone (1000 ml)
was
stirred in a 4-necked flask with mechanical stirrer, thermometer and dripping
funnel.
Then a solution of 2-bromo-1-(3-nitrophenyl)ethanone (1.93 mol) in 2-propanone
(2500 ml) was added dropwise while cooling on an ice bath to keep the
temperature
below 20 C. The reaction mixture was stirred overnight. The resulting
precipitate was
filtered off, washed with 2-propanone and dried, yielding 640 g of
intermediate (2).
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HO~S
b) Preparation of o2N Njintermediate (3)
A suspension of intermediate (2) (1.85 mol) in ethanol (3000 ml) was chilled
on an ice
bath. Sodium borohydride (2.78 mol) was added portionwise and the reaction
mixture
was allowed to warm to room temperature overnight. A saturated NaHCO3 solution
(1000 ml) and water (500 ml) were added and after quenching dichloromethane
was
added. The resulting mixture was stirred for 1 hour and the aqueous layer was
extracted with dichloromethane (4 x 1000 ml). The organic layers were
combined,
dried, filtered off and the solvent was evaporated, yielding 469 g of
intermediate (3).
~
c) Preparation of IN intermediate (4)
02N
A suspension of intermediate (3) (0.34 mol) in 1,2-dichloroethane (3000 ml)
was
stirred at room temperature and a solution of thionyl chloride (0.68 mol) in
1,2-dichloroethane (150 ml) was added dropwise over 6 hours. The reaction
mixture
was stirred overnight and NaHCO3 (1500 ml) was added carefully. The reaction
mixture was warmed to 40 C, stirred for 6 hours and then the aqueous layer was
removed. The organic layer was washed with a saturated NaHCO3 solution (4 x
1000
ml), dried, filtered off and the solvent was evaporated. The obtained residue
was
dissolved in CH2C12/CH3OH (95/5; 2000 ml) and stirred with silica gel (250 g).
The
silica gel was filtered off. The filtrate was filtered again over silica gel
(1000 g) and
the solvent was evaporated, yielding 253 g of intermediate (4).
lu,,. d) Preparation of fl<NS
(S) \,-N-,> intermediate (5)
02N
Intermediate (4) was separated into its enantiomers by chiral column
chromatography
over Chiralcel AS 1000A 20 m (eluent : ethanol/heptane 30/70). The desired
fractions comprising the (S)-enatiomer were collected and the solvent was
evaporated,
yielding (6S)-(3-nitro-phenyl)-2,3,5,6-tetrahydro-imidazo[2,1-b]thiazole
(interm. 5).
The fractions comprising the (R)-enantiomer were also collected and, after
evaporation
of the solvent, yielded (6R)-(3-nitro-phenyl)-2,3,5,6-tetrahydro-imidazo[2,1-
b]thiazole
(interm. 6).
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Example A.3
Preparation of fl<NS
(S) ~N~ intermediate (7)
H2N
a) A solution of intermediate (5) (0.15 mol) in ethyl acetate was stirred at
room
temperature. A 1M solution of hydrochloric acid (0.199 mol) in diethylether
(190 ml)
was added dropwise. The resulting precipitate was filtered off and dried at
low
pressure, yielding 39.4 g of the hydrochloric acid salt of intermediate (5).
b) Water (105 ml) and then methanol (35 ml) were added to a mixture of the
hydrochloric acid salt of intermediate (5) (0.0122 mol), iron powder (0.061
mol) and
ammonium chloride (0.061 mol). The reaction mixture was stirred and warmed to
70 C for 30 minutes and was then allowed to cool to room temperature. 0.1N
HC1(14
ml) was added and the resulting mixture was filtered over Kieselguhr. The
Kieselguhr
was washed with 0.O1N HC1(175 ml) and with dichloromethane (175 ml). The
obtained filtrate was stirred while a saturated NaHCO3 solution (175 ml) and
NaHCO3
(10 g) were added. The organic layer was separated and the aqueous layer was
extracted with dichloromethane (4 x 175 ml). The organic layers were combined,
dried, filtered off and the solvent was evaporated, yielding 2.61 g of
intermediate (7).
Using an analogous procedure but starting from intermediate (4), 3-(2,3,5,6-
tetrahydro-
imidazo[2,1-b]thiazol-6-yl)-phenylamine was prepared as intermediate (8).
intermediate (8)
H2N
Example A.4
H
Preparation of p intermediate (9)
0
At -60 C, a solution of dimethylsulfoxide (36.5 ml, 0.514 mol) in
dichloromethane
(700 ml) was added dropwise to a 2M solution of oxalyl chloride (26.9 ml,
0.308 mol)
in dichloromethane. The mixture was stirred for 30 minutes and then a solution
of
tetrahydrofurfuryl alcohol (25 ml, 0.257 mol) in dichloromethane (100 ml) was
added
dropwise. The mixture was stirred for 20 minutes and then triethylamine (181
ml, 1.29
mol) was slowly added. The reaction mixture was warmed up to room temperature
and
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stirred for 30 minutes. The reaction mixture was filtered off and the residue
was
washed with dichloromethane (250 ml). The combined dichloromethane layers were
washed with water (150 ml), dried and evaporated carefully at 25 C. To the
residue
was added diethyl ether, the precipitate was filtered off and washed with
diethyl ether.
The combined organic layers were evaporated carefully. The product was
purified by
bulb-to-bulb distillation (75 C, 20 mbar) yielding 12.6 gram of intermediate
(9).
B. Preparation of the fmal compounds
Example B.1
Y
IIuS~NJ
Preparation of ()HN 0 compound (1)
~Ilu~~
O ~0
A 4M solution of hydrochloric acid in dioxane (16.43 ml) was added dropwise to
a
solution of intermediate (7) (0.066 mol) in anhydrous acetonitrile (500 ml).
The
resulting mixture was chilled on an ice-bath. A mixture of intermediate (1)
(0.069 mol)
in acetonitrile (100 ml) was added dropwise and the reaction mixture was
allowed to
reach room temperature overnight. Acetonitrile was evaporated. A saturated
NaHCO3
solution (500 ml) was added and the resulting mixture was extracted with
dichloro-
methane (3 x 500 ml). The organic layers were combined, dried, filtered off
and the
solvent was evaporated. The residue was purified by flash column
chromatography
(eluent: CH2C12/CH3OH 95/5) over silica gel. The product fractions were
collected and
the solvent was evaporated, yielding 12.23 g of compound (1) (mp. 42-57 C)
(specific
optical rotation OR =-3.77 (589 nm, c = 0.4636 w/v%, methanol, 20 C)).
Using the procedure as outlined above, the compounds (2), (3) and (4) were
also
prepared using respectively a combination of intermediate (8) and racemic
tetrahydro-
2-furancarbonyl chloride, or intermediate (7) and racemic tetrahydro-2-
furancarbonyl
chloride, or intermediate (7) and (2S)-tetrahydro-2-furancarbonyl chloride, as
the
starting materials.
N~S, N
~ 0 compound (2) ~ 0 compound (4)
0~~ 0~~
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~
HN ~ compound (3)
0~-G
Example B.2
Preparation of
~ ~ compound (A)
0
\01
Intermediate (7) (470 mg) was dissolved in anhydrous acetonitrile (40 ml). A
solution
of 4 M HC1 in dioxane (0.538 ml) was added dropwise. The mixture was cooled
using
an ice bath and a solution of 2-furancarbonyl chloride (0.213 ml) in
acetonitrile (20 ml)
was added dropwise. The reaction mixture was heated at 40 C overnight. An
aqueous
saturated NaHCO3 solution (150 ml) was added to the reaction mixture. The
reaction
mixture was extracted three times with dichloromethane (150 ml) and the
combined
organic layers were dried, filtered and the solvent was removed by
evaporation. The
residue was isolated and purified using preparative thin layer chromatography
with a
mixture of dichloromethane/methanol (95:5) as eluent, yielding 210 mg of
compound
(A) (mp. 77 C).
This compound (A) is known from US-4,014,892 as compound (163).
Example B.3
Y>
IIuS~N~/
Preparation of ~ ~
HN 0 compound (B)
\-/O
A solution of intermediate (7) (550 mg) was stirred in dry acetonitrile (30
ml) and
NaHCO3 (181 mg) was added. A solution of intermediate (9) in dry acetonitrile
(15 ml) was added and after 15 minutes an amount of NaBH(OAc)3 (547 mg) was
added as a solid. After 2 hours TLC revealed that the reaction was complete.
Saturated
aqueous NaHCO3 was added and the product was extracted twice with ethyl
acetate.
The combined ethyl acetate layers were dried and evaporated. The resulting
residue
was purified with flash column chromatography on silica using a mixture of
dichloromethane and methanol in the following ratio and elution times : 10
minutes
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3% methanol; 20 minutes 3% methanol to 5% methanol; 20 minutes 5% methanol
with
a flow rate of 30 ml/minute, yielding 237 mg of compound (B).
This compound (B) is known from US-4,014,892 as compound (163) and from
GB-1,365,515 as compound (81).
C. Pharmacological examples
C.1. Efficacy study of anthelmintics in in vivo H. contortus/jirds model
The anthelmintic efficacy of the compounds of the present invention was
evaluated in
an in vivo model using unmedicated jirds (Meriones unguiculatus), inoculated
three
times with approximately 300 exsheated infective larvae of Haemonchus
contortus
(multiresistant strain), treated orally with a test compound 11 days after
their first
infection with H. contortus larvae, and necropsied on day 14 to count the
number of
recovered H. contortus worms. The anthelmintic efficacy of the art known
compounds
(A) and (B) was also evaluated using the same model.
Animals
Female CRW jirds aged between 28 and 35 days and weighing 30-35 g (Charles
River,
Sulzfeld, Germany) were used. Three jirds each were assigned randomly upon
arrival
to translucent polysulfone individually ventilated cages (48 x 37.5 x 21 cm)
containing
wood shavings. Commercial rodent chow and water were given ad libitum.
Following a
four-day acclimation period, the jirds were artificially infected.
Parasite
A PolyRes strain of Haemonchus contortus (resistant against levamisole,
mebendazole,
ivermectin and closantel) was used. This strain has been maintained in
artificially
infected male donor lambs. Individual feces containing Haemonchus eggs were
collected in fecal bags. The fecal pellets were broken, mixed with charcoal,
moistened
and put in an incubator for embryonation at 28 C and 95 % relative humidity.
Seven
days later this mixture was placed in Baermann funnels and third stage
ensheathed
larvae were collected after 12 hours. These larvae were rinsed with water for
cleaning
and disinfected with a 2 % formalin solution. Such larvae can be used
immediately for
artificial infection or can be stored in the fridge at about 8 C for a maximum
duration
of 6 months. Infective larvae (< 6 months old) were exsheathed by rinsing with
a
3.3 vol % commercial sodium hypochlorite solution during 10 minutes, filtered
through
a Buchner funnel, rinsed with water, concentrated in a Baermann funnel, and
collected
after 2 hours. Exsheathed larvae prepared in this manner can be used for
subsequent
infection of jirds or laid in a supply for extended periods by cooling in the
gas over
liquid nitrogen during 1 hour and storing in liquid nitrogen at -196 C.
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Infections
All jirds were inoculated orally with approximately 300 exsheathed infective
larvae of
H. contortus per dose on three consecutive days. Inoculations were
administered using
a blunted 18 G dosing needle fitted to a 1 mi syringe.
Treatments
Eleven days after their first infection jirds were treated with test
compounds, suspended
or dissolved in 0.4 ml DMSO, and the dose to be tested was administered in a
volume
of 0.1 ml / 50 g bodyweight via a blunted 18 G dosing needle fitted to a 1 mi
syringe.
Control animals, included in each experiment, remained untreated. Levamisole
hydrochloride, mebendazole, ivermectin and closantel were used in different
dosage
titration experiments to validate the model.
Necropsy
All jirds were starved 20 hours before necropsy and killed on day 14 past
first infection
by CO2 inhalation. For worm recovery their stomachs were removed, opened
longitudinally, and incubated in a beaker with 20 ml digestion fluid (10 g
pepsin +
30 ml concentrated hydrochloric acid) at 37 C for 3 hours. Following
digestion, the
stomach content was passed through a tea-strainer, the passage fluid was
catched in a
sieve (32 ,um) and the worms were recovered with tap water. The beakers were
stored
in the fridge for subsequent counting.
Examination and percentage efficacy
The content of each beaker was mixed, poured over in a 6-well plate in 6
aliquots, and
the worms were counted under an inverted microscope. The percentage efficacy
for
each test compound was determined and the results are summarized in Table 1
below.
Table 1: efficacy data in clearing PolyRes strain Haemonchus contortus from
jirds
after oral treatment with a test compound
Test Compound Dose mpk No. of Animals Med. W.b. % Efficacy
Untreated Controls 0 36 30.0 -
Co. No. 1 2.5 3 36.0 65.9
Co. No. 1 5 3 6.0 79.3
Co. No. 1 10 6 0.0 100.0
Co. No. 4 5 3 5.0 68.8
Co. No. 4 10 3 3.0 85.0
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Test Compound Dose mpk No. of Animals Med. W.b. % Efficacy
Co. No. A 5 3 11.0 35.3
Co. No. A 10 3 7.0 65.0
Co. No. B 5 6 10.0 55.6
Co. No. B 10 3 14.0 60.6
Dose mpk : dose of the test compound in mg per kg body weight
No. of Animals : number of test animals
Med. W.b. : median worm burden
Percentage efficacy =({(mean number of worms recovered from control group) -
(mean number of worms recovered from treated group)} divided
by (mean number of worms recovered from control group)) x 100
As can be seen in Table 1 both compounds (1) and (4) have a better
demonstrated
anthelmintic efficacy than the art known compounds (A) and (B).
